DESCRIPTION: One of the major challenges limiting the clinical use of large, tissue engineered grafts has been the inability to provide adequate oxygen supply to facilitate the post-implantation survival of cells during the period of vascular in-growth and tissue integration. As a consequence, cells rely solely on limited oxygen diffusion from surrounding tissue. The inner regions of engineered graft rapidly become hypoxic leading to massive cell death, the formation of necrotic cores, and failure of the graft. To overcome these limitations, it is imperative to develop novel methods to provide sustained, localized oxygen delivery to maintain the viability of cells during the critical period between the implantation of the graft and adequate invasion of host vasculature bringing oxygenated blood. We propose an innovative approach utilizing hollow polymeric microspheres, which have the appropriate material properties to facilitate oxygen loading at elevated pressures and controlled release along the pressure gradient into the immediate microenvironment. By varying the size of the microspheres, the intrinsic oxygen permeability and thickness of the polymer shell, and pressure to which these micro tanks are filled, we propose to tune the amount, rate, and duration of oxygen delivery to cells embedded in a hydrogel or biomaterial scaffold. This versatile, innovative technique is unprecedented in its potential for exquisite spatiotemporal control of oxygen delivery for therapeutic application in tissue engineering and regenerative medicine. In this study, we will obtain commercially available micro tanks made of polyvinylidene chloride (PVDC) and poly acrylonitrile (PAN) and sort into two different sizes (38 - 45 ?m & 75 - 90 ?m) and shell thicknesses (2 ? 0.5 ?m & 8 ? 2 ?m). We hypothesize that we can tune the oxygen delivery to maintain cellular viability and biological properties using these groups of micro tanks. The objective of the application is to generate proof- of-concept data by testing this hypothesis in two Specific Aims. In Sp. Aim 1, we will characterize these eight groups of micro tanks to determine rupture strength and the oxygen release profiles over time. In Sp. Aim 2, we will assess the potential for using these micro tanks to provide sustained oxygen delivery to adipose derived stem cells (ASCs) cultured in anoxic (0% O2) or hypoxic (2% O2) environments for up to two weeks in vitro. Subsequently, we will test the potential to provide appropriate oxygen delivery to chondrocytes, ASCs, and adipocytes: three cell phenotypes, which were specifically selected to have orders of magnitude differences in specific oxygen uptake rates (OURs). We will assess cell viability, proliferation, gene expression (for cell survival and apoptotic genes) and the generation of reactive oxygen species (ROS), which indicate oxygen toxicity. These proof-of-concept studies will formulate the foundation for future development of biodegradable micro tanks and transplantation of tissue engineered grafts to in vivo defect sites in small animals.